This invention relates to a pharmaceutical composition and method of inhibiting human immunodeficiency virus (HIV) and, more particularly, to a synergistic combination of the N-butyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol (deoxynojirimycin) and 3'-azido-3'-deoxythymidine (AZT) in amount which achieves antiviral efficacy, having potential use for the treatment of acquired immune deficiency syndrome (AIDS) and AIDS related complex (ARC).
Acquired immune deficiency syndrome, which only a few years ago was a medical curiosity, is now a serious disease. As a consequence, a great effort is being made to develop drugs and vaccines to combat AIDS. The AIDS virus, first identified in 1983, has been described by several names and has the capacity to replicate within cells of the immune system and thereby lead to a profound destruction of T4. T-cells (or CD4.sup.+ cells). See, e.g., Gallo et al., Science 224, 500-03 (1984), and Popovic et al., Ibid., 497-500 (1984).
This virus, which has been established to be a retrovirus, had been known as lymphadenopathy-associated virus (LAV) or AIDS-related virus (ARV). Two distinct AIDS viruses, HIV-1 and HIV-2, have been described. HIV-1 is the virus originally identified in 1983 by Montagnier and co-workers at the Pasteur Institute in Paris [Ann. Virol. Inst. Pasteur 135 E, 119-134 (1984)], while HIV-2 was more recently isolated by Montagnier and his coworkers in 1986 [Nature 326, 662 (1987)]. As used herein, HIV is meant to refer to these viruses in a generic sense.
Although the molecular biology of AIDS is beginning to be unraveled and defined, much more needs to be learned and understood about this disease. In the meantime, numerous approaches are being investigated in the search for potential anti-AIDS drugs and vaccines. Development of an AIDS vaccine is hampered by lack of understanding of mechanisms of protective immunity against HIV, the magnitude of genetic variation of the virus, and the lack of effective animal models for HIV infection. See, for example, Koff and Hoth, Science 241, 426-432 (1988).
One approach to designing drugs or VaCcines for treating human beings infected by HIV depends on the knowledge molecular biologists have acquired on the replicative life cycle of the virus as it enters and infects a host cell, replicates, and goes on to infect other cells. Within the replicative cycle of HIV there are certain virus-specific steps which are potential targets for antiviral therapy. Hirsch et al., Antimicrob. Agents Chemother. 31, 839-43 (1987).
The first drug to be approved by the U.S. Food and Drug Administration (FDA) for treatment of AIDS was zidovudine, better known under its former name azidothymidine (AZT). Chemically, this drug is 3'-azido-3'-deoxythymidine, and has the following structure: ##STR1##
AZT is converted by cellular kinases to a triphosphate form which is a strong competitive inhibitor of HIV reverse transcriptase, thus interfering with an early stage in HIV replication.
This drug was originally selected as a potential weapon against AIDS because it was shown to inhibit replication of the virus in vitro. Such tests are virtually the only practical method of initially screening and testing potential anti-AIDS drugs.
Although AZT is now used clinically, a serious drawback of AZT is its toxic side-effects. Indeed, the FDA required package insert for Retrovir.RTM., the commercially available pharmaceutical composition containing AZT as the active ingredient, warns of hematologic toxicity including granulocytopenia and severe anemia requiring transfusions. Thus, the search for better anti-AIDS drugs continues.
More recently, certain glucosidase inhibitors have been tested for activity against the AIDS virus. Because the envelope glycoproteins of HIV are heavily glycosylated, compounds that interfere with co- and postranslational processing of glycoprotein gp120 and the transmembrane glycoprotein gp41 may prevent viral entry into a cell. Karpas et al., Proc. Natl. Acad. Sci. USA 85, 9229-9233 (1988). These compounds interfere, or interrupt a different stage in the HIV replicative process than AZT.
Three such compounds suggested as potential anti-AIDS drugs are castanospermine, 1-deoxynojirimycin (DNJ) and 2,5-dihydroxymethyl-3,4-dihydroxy-pyrrolidine (DMDP). See, e.g., Sunkara et al., Biochem. Bioohvs. Res. Commun. 148(1), 206-210 (1987); Tyms et al., Lancet, Oct. 31, 1987, pp. 1025-1026; Walker et al., Proc. Natl. Acad. Sci. USA 84, 8120-8124 (1987); and Gruters et al., Nature 330, 74-77 (1987).
Thus, castanospermine, which is an alkaloid isolated from the seeds of Australian chestnut tree, has been found to interfere with normal glycosylation of HIV virions, thereby altering the envelope glycoprotein and preventing entry of HIV into target cells. However, since castanospermine is in limited supply due to its natural source, it is extremely expensive, and therefore not a realistic candidate for a drug urgently required on a large scale. Additionally, castanospermine has demonstrated cytotoxicity at dose levels of 0.70 mg/ml. Karpas et al., Proc. Nat'l. Acad. Sci. USA 85, 9229-9233 (1988).
In PCT Inter. Appln. WO 87/03903, published July 2, 1987, the N-methyl derivative of deoxynojirimycin (DNJ) also was disclosed as having activity against HIV ostensibly based on its glucosidase I inhibitory activity. However, it was subsequently shown by Fleet et al., FEBS Lett 237, 128-132 (1988), that not all glucosidase I inhibitors are effective inhibitors of HIV. Therefore, some other mechanism may be responsible for HIV inhibitory activity.
The N-butyl derivative of deoxynojirimycin (N-butyl DNJ) has been found to have enhanced inhibitory activity against the human immunodeficiency virus (HIV) at non-toxic concentrations compared to that exhibited by the corresponding N-methyl and N-ethyl derivatives.
N-butyl DNJ has the following chemical structure: ##STR2## In order to indicate stereoisomerism, solid and dotted lines show bonds directed above or below, respectively, the plane of the paper.
N-butyl DNJ uniquely reduces the virus titer by over five logs at non-cytotoxic concentrations whereas the N-methyl- and N-ethyl-deoxynojirimycin derivatives cause only a two to four log-order of reduction in the yield of infectious HIV. As such, the N-butyl derivative has significant potential use for the treatment of acquired immune deficiency syndrome (AIDS), and phase I clinical trials of this agent have recently been announced.
Despite the above noted advances made in drug therapy for HIV infected individuals, it is believed that effective non-toxic therapy directed against HIV may require a chemotherapeutic approach which involves the use of combination therapies. Such an approach has not been widely employed in anti-HIV therapy, but has been successful in the treatment of a variety of bacterial and fungal infections, as well as in cancer chemotherapy. Combinations of anti-HIV therapies which include agents that attack the HIV replicative cycle at multiple sites offer several advantages, particularly if favorable drug interactions occur.
When two agents are combined, they may have one of three types of activity against HIV replication in vitro, as established by the Combination Index (CI) discussed hereinafter.
1. Additive effect: Two drugs are said to be additive when the activity of the drugs in combination is equal to the sum (or a partial sum) of their independent activities when studied separately.
2. Synergism: The combined effect of a synergistic pair of agents is greater than the sum of their independent activities when measured separately.
3. Antagonism: If two drugs are antagonistic, the activity of the combination is less than the sum of their independent effects when measured alone.
The goals of combination therapy should include the ability to target different sites in the HIV replicative cycle, and to affect viral replication in a broad range of cell types. Also, the agents should not display additive toxicity in combination. The benefits of combination therapy include potentially additive or synergistic interactions in vitro, which may allow the use of individual drugs below their toxic concentrations. Also, combination therapy may prevent the emergence of drug-resistant HIV mutants.
Studies combining various known anti-HIV agents have already been conducted. For example, combinations of either AZT or 2',3'-dideoxycytidine and recombinant alpha-A interferon have been shown to inhibit HIV synergistically in vitro. Hartshorn et al., Antimicrob. Ag. Chemother. 31, 168-72 (1987); Vogt et al., J. Infect. Dis. 158, 378-85 (1988). Other combinations, such as AZT plus acyclovir have shown synergism. Mitsuya et al. in S. Broder (ed.) AIDS, Modern Concepts and Therapeutic Challenges, Marcel Dekker, Inc., N.Y. However, other tested drug combinations such as AZT and ribavirin (1,.beta.-D-ribofuranosyl-11-1,2-triazole-3-carboxamide) have been shown to be antagonistic in vitro. Vogt et al., Science 23, 1376-79 (1987).